ABSTRACT
The nucleolus is the site of ribosome biosynthesis encompassing the ribosomal DNA (rDNA) locus in a phase separated state within the nucleus. In budding yeast, we find the rDNA locus and Cdc14, a protein phosphatase that co-localizes with the rDNA, behave like a condensate formed by polymer-polymer phase separation, while ribonucleoproteins behave like a condensate formed by liquid-liquid phase separation. The compaction of the rDNA and Cdc14's nucleolar distribution are dependent on the concentration of DNA cross-linkers. In contrast, ribonucleoprotein nucleolar distribution is independent of the concentration of DNA cross-linkers and resembles droplets in vivo upon replacement of the endogenous rDNA locus with high-copy plasmids. When ribosomal RNA is transcribed from the plasmids by Pol II, the rDNA-binding proteins and ribonucleoprotein signals are weakly correlated, but upon repression of transcription, ribonucleoproteins form a single, stable droplet that excludes rDNA-binding proteins from its center. Degradation of RNA-DNA hybrid structures, known as R-loops, by overexpression of RNase H1 results in the physical exclusion of the rDNA locus from the nucleolar center. Thus, the rDNA locus is a polymer-polymer phase separated condensate that relies on transcription and physical contact with RNA transcripts to remain encapsulated within the nucleolus.
Subject(s)
Cell Cycle Proteins/metabolism , Cell Cycle , Cell Nucleolus/metabolism , DNA, Ribosomal/metabolism , Protein Tyrosine Phosphatases/metabolism , R-Loop Structures , RNA Polymerase I/metabolism , Ribonucleoproteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Cycle Proteins/genetics , Cell Nucleolus/genetics , Clinical Trials, Phase I as Topic , DNA, Ribosomal/genetics , G1 Phase/drug effects , G1 Phase/genetics , G2 Phase Cell Cycle Checkpoints/drug effects , G2 Phase Cell Cycle Checkpoints/genetics , Hydro-Lyases/metabolism , Kinetics , Microtubule-Associated Proteins/metabolism , Nuclear Proteins/metabolism , Polymers/chemistry , Polymers/metabolism , Protein Tyrosine Phosphatases/genetics , RNA Polymerase I/genetics , Ribonuclease H/genetics , Ribonuclease H/metabolism , Ribonucleoproteins/genetics , Ribonucleoproteins, Small Nuclear/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Sirolimus/pharmacology , Up-Regulation , Water/chemistry , Water/metabolismABSTRACT
Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.
Subject(s)
Ear/anatomy & histology , Fibroblasts/cytology , Primary Cell Culture/methods , Tail/cytology , Animals , Cell Line , Mice , Mice, Inbred C57BLABSTRACT
The DNA damage response (DDR) alerts the immune system to the danger posed by DNA damage through the induction of damage-associated molecular pattern molecules, chemokines, and ligands for activating immune receptors such as lymphocyte function-associated antigen 1 (LFA-1), NKG2D, and DNAX accessory molecule 1 (DNAM-1). Here we provide evidence that OVA(257-264) -pulsed fibroblasts gain the ability to activate naïve OT-I CD8(+) T cells in response to DNA damage. The ability of fibroblasts to activate OT-I CD8(+) T cells depended on the upregulation of ICAM-1 on fibroblasts and DNAM-1 expression of CD8(+) T cells. OVA(257-264) -pulsed fibroblasts were able to induce a protective T-cell response against B16-OVA cells in a DDR-dependent manner. Hence, the DDR may alert the immune system to the presence of potentially dangerous cells by upregulating the expression of ligands that can induce the activation of innate and adaptive immune cells.
